The present invention generally relates to a method and apparatus for measuring the velocity of an object, and more specifically, to sensing light from an object with a light sensitive detector, the amplitude of that light signal having been modulated by an optical grating, and measuring the velocity of the object by analysis of the modulated light signal.
Cells and cell groupings are three-dimensional objects containing rich spatial information. The distribution of a tremendous variety of bio-molecules can be identified within a cell using an ever-increasing number of probes. In the post-genome era there is mounting interest in understanding the cell, not only as a static structure, but as a dynamic combination of numerous interacting feedback control systems. This understanding can lead to new drugs, better diagnostics, more effective therapies, and better health care management strategies. However, this understanding will require the ability to extract a far greater amount of information from cells than is currently possible.
The principal technologies for cellular analysis are automated microscopy and flow cytometry. The information generated by these mature technologies, although useful, is often not as detailed as desired. Automated microscopy allows two-dimensional (2D) imaging of from one to three colors of cells on slides. Typical video frame rates limit kinetic studies to time intervals of 30 ms.
Instruments known as flow cytometers currently provide vital information for clinical medicine and biomedical research by performing optical measurements on cells in liquid suspension. Whole blood, fractionated components of blood, suspensions of cells from biopsy specimens and from cell cultures, and suspensions of proteins and nucleic acid chains are some of the candidates suitable for analysis by flow cytometry. In flow cytometers specialized for routine blood sample analysis, cell type classification is performed by measuring the angular distribution of light scattered by the cells and the absorption of light by specially treated and stained cells. The approximate numbers of red blood cells, white blood cells of several types, and platelets are reported as the differential blood count. Some blood-related disorders can be detected as shifts in optical characteristics, as compared to baseline optical characteristics, such shifts being indicative of morphological and histochemical cell abnormalities. Flow cytometers have been adapted for use with fluorescent antibody probes, which attach themselves to specific protein targets, and for use with fluorescent nucleic acid probes, which bind to specific DNA and RNA base sequences by hybridization. Such probes find application in medicine for the detection and categorization of leukemia, for example, in biomedical research, and drug discovery. By employing such prior art techniques, flow cytometry can measure four to ten colors from living cells. However, such prior art flow cytometry offers little spatial resolution, and no ability to study a cell over time. There is clearly a motivation to address the limitations of existing cell analysis technologies with a novel platform for high speed, high sensitivity cell imaging.
A key issue that arises in cell analysis carried out with imaging systems is the measurement of the velocity of a cell or other object through the imaging system. In a conventional time-domain methodology, cell velocity is measured using time-of-flight (TOF). Two detectors are spaced a known distance apart and a clock measures the time it takes a cell to traverse the two detectors. The accuracy of a TOF measurement is enhanced by increasing detector spacing. However, this increases the likelihood that multiple cells will occupy the measurement region, requiring multiple timers to simultaneously track all cells in view. Initially, the region between the detectors is cleared before starting sample flow. As cells enter the measurement region, each entry signal is timed separately. The system is synchronized with the sample by noting the number of entry signals that occur before the first exit signal.
TOF velocity measurement systems are prone to desynchronization when the entry and exit signals are near threshold, noise is present, or expected waveform. characteristics change due to the presence of different cell types and orientations. Desynchronization causes errors in velocity measurement which can lead to degraded signals and misdiagnosed cells until the desynchronized condition is detected and corrected. Resynchronization may require that all cells be cleared from the region between the detectors before restarting sample flow, causing the loss of sample.
Significant advancements in the art of flow cytometry are described in commonly assigned U.S. Pat. No. 6,249,341, issued on Jun. 19, 2001, and entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS, as well as in commonly assigned U.S. Pat. No. 6,211,955, issued on Apr. 3, 2001, also entitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS. The specifications and drawings of each of these patents are hereby specifically incorporated herein by reference.
The inventions disclosed in the above noted patents perform high resolution, high-sensitivity two-dimensional (2D) and three-dimensional (3D) imaging using time-delay-integration (TDI) electronic image acquisition with cells in flow. These instruments are designed to expand the analysis of biological specimens in fluid suspensions beyond the limits of conventional flow cytometers. TDI sensors utilize solid-state photon detectors such as charge-coupled device (CCD) arrays and shift lines of photon-induced charge in synchronization with the flow of the specimen. The method allows a long exposure time to increase a signal-to-noise ratio (SNR) in the image while avoiding blurring. However, precise synchronization of the TDI detector timing with the motion of the moving targets is required. For example, if a target is to traverse 100 lines of a TDI sensor to build an image, and the blurring is expected to be less than a single line width, then the velocity of the target must be known to less than one percent of its actual value. It would thus be desirable to provide method and apparatus capable of producing highly accurate flow velocity for such moving targets.
Several methods have been suggested in the prior art to address the limitations of TOF velocity measurements, and to achieve highly accurate flow velocity for moving targets such as cells. One such technique is laser Doppler anemometry (LDA), in which one or more laser beams are used to interrogate a moving target. The Doppler frequency shift is detected as modulation from the interference of multiple beams that have traversed different paths in the apparatus. An example of such an apparatus is disclosed in U.S. Pat. No. 3,832,059, issued on Aug. 27, 1974, and entitled FLOW VELOCITY MEASURING ARRANGEMENT UTILIZING LASER DOPPLER PROBE. That apparatus employs two laser beams that are directed toward a moving target at converging angles. The back-scattered light from both beams is collected and focused on a common photodetector. Coherent interference at the detector generates modulation with frequency equal to twice the Doppler shift frequency, allowing the detection of the target velocity. While LDA is a functional technique, the LDA systems described in U.S. Pat. No. 3,832,059 are elaborate, expensive, and prone to instability.
In an attempt to overcome the problem of instabilities in the laser wavelength, an improved LDA apparatus is disclosed in U.S. Pat. No. 5,229,830, issued on Jul. 20, 1993, and entitled DOPPLER VELOCIMETER. In this improved apparatus, a rotating grating is added for the purpose of extracting diffraction side-lobes for use in interrogating the target. This approach eliminates the wavelength dependency in the velocity measurement, but adds even more cost and complexity to the velocimeter.
An alternative approach to measuring object velocity by coherent light interference is found in LDA instruments that generate fringe patterns in the measurement field. Multiple laser beams create either stationary or propagating fringe patterns with known periods. Objects crossing the fringes modulate the light collected by the instruments. The modulation frequency is equal to the velocity divided by the fringe pattern period. LDA instruments of this type are disclosed in U.S. Pat. No. 4,148,585, issued on Apr. 10, 1979, and entitled THREE DIMENSIONAL LASER DOPPLER VELOCIMETER; and in U.S. Pat. No. 5,160,976, issued on Nov. 3, 1992, and entitled OPTICAL DETERMINATION OF VELOCITY USING CROSSED INTERFERENCE FRINGE PATTERNS. The apparatus disclosed in the first of these two patents uses a rotating diffraction grating and a beam-splitter to generate four interrogation beams, which are then crossed in various combinations to measure velocity as a (3D) vector. The apparatus disclosed in U.S. Pat. No. 5,160,976 uses specially constructed optical fiber bundles and two lasers of differing wavelength to generate two fringe patterns at the target, such that the patterns are oriented perpendicular to one another, in order to measure velocity as a 2D vector. Such fringe-pattern velocimeters have been adapted for use in mapping complex flow fields. However, the cost, complexity, and instability inherent in delivering multiple coherent beams to the sample volume, with tightly constrained wavelengths and alignments, is a deterrent to their use. It would be desirable to provide an alternative method and apparatus to measure the velocity of an object entrained in a flow of fluid which is less costly, and more forgiving than these prior solutions to this problem.
The LDA systems described above rely on the mechanisms of Doppler shift and wave interference for the measurement of velocity. An alternative approach to measuring the velocity of objects in fluids is to collect data in short bursts at carefully timed intervals, i.e., to collect stroboscopic snapshots of the objects. This approach, which is related to but morescomplicated than the TOF method described above, is called object imaging velocimetry (PIV). One such system is disclosed in U.S. Pat. No. 4,729,109, issued on Mar. 1, 1988, and entitled METHOD AND APPARATUS FOR MEASURING THE DISPLACEMENTS OF PARTICLE IMAGES FOR MULTIPLE EXPOSURE VELOCIMETRY. In this patent, a strobe light source illuminates the target field. Cylindrical lenses collapse the image of the field into two orthogonal linear projections which are captured by linear arrays of photodetectors. Signals are collected from the detector arrays for every flash of the strobe light. For each axis, the signal from a first exposure is correlated with the signal from the subsequent exposure to measure target displacement. The displacement is converted to velocity by dividing the displacement by the time between exposures. The apparatus forms images in which contrast is created by light absorption by the target.
A PIV instrument utilizing target fluorescence is disclosed in U.S. Pat. No. 5,333,044, issued on Jul. 26, 1994, and entitled FLUORESCENT IMAGE TRACKING VELOCIMETER. In the apparatus described in this patent, a planar region in the field of flow is illuminated using a strobe-coherent light source. A detector forms 2D images of the fluorescent emission of objects stained with a fluorescent dye. Computer analysis of the sequence of captured images is used to track the motion of objects in the plane of illumination.
It should be noted that the stabilization and alignment of PIV systems are less problematic than in LDA systems, but the PIV systems require pulsed illumination or gated data acquisition to establish timing. The PIV systems also require arrays of detectors and elaborate data analysis to yield velocity measurements.
A technology that provides a simple, cost-effective alternative to LDA and PIV for measuring object velocity in fluids is based on the insertion of a grating with alternating opaque and transparent parallel bars in the light path of the photo-sensor. Light from moving objects is modulated by the optical grating pattern to create a signal with frequency directly proportional to the component of velocity perpendicular to the optical grating bars. If object motion is constrained to this perpendicular direction, then the frequency is equal to the true velocity divided by the period, or pitch, of the optical grating. A laser velocimeter based on this principle for measuring the velocity of a reflective surface moving relative to the instrument is disclosed in U.S. Pat. No. 3,432,237, issued on Mar. 11, 1969, and entitled VELOCITY MEASURING DEVICE. In the disclosed system of this patent, the target surface is illuminated with a continuous wave laser and light scattered by the moving surface is collected by a lens and then delivered to a photosensitive detector through a grating. The bars of the optical grating are oriented perpendicular to the axis of motion. An electronic frequency measuring circuit is used to determine the frequency of the photosensitive detector. The frequency is conveyed directly to a display device for viewing and conversion to velocity.
The application of this method to objects suspended in fluid is disclosed in U.S. Pat. No. 3,953,126, issued on Apr. 27, 1976, and entitled OPTICAL CONVOLUTION VELOCIMETER. In the disclosed apparatus of this patent, light collimated by a lens passes through the flow of fluid and is reflected by a mirror with alternating bars of reflective and absorptive material. The reflective bars return light through the flow of fluid to be collected by the lens. The lens focuses the reflected light on a photosensitive detector. An electronic circuit is used to estimate the frequency of the detector signal and to deliver the frequency to a display device for viewing.
It should be noted that the hardware signal processors used in early implementations of laser velocimeters have largely been displaced by computation-based digital signal processors. The demands on the photosensor signal processors vary with the nature of the application, but the most stringent applications demand high speed and high accuracy, under conditions of low SNR and rapidly varying flow velocity.
An example of an effective method for extracting velocity from the photosensor signal of a grating-based laser velocimeter is disclosed in U.S. Pat. No. 5,859,694, issued on Jan. 12, 1999, and entitled OPTICAL VELOCIMETER PROBE. In this patent, the digitized photosensor signal is captured in blocks of samples for processing. For each block, the signal processor executes the steps of generating a complex signal using the Hilbert transform, autocorrelating the complex signal, and extracting the phase for each time sample of the auto-correlogram. The autocorrelation is performed using the steps of a complex Fourier transformation, squaring the magnitude of the spectrum, and then applying an inverse Fourier transformation. Finally, an optimization routine finds a best-fit velocity value for the phase samples. The method described in this patent has the advantage of building SNR and delivering accurate velocity estimates, given long signal segments. However, the method is computation intensive, limiting the rate at which the velocity estimate is updated.
It would be desirable to utilize the principal of modulation of light from moving objects by the insertion of a periodic grating into the detector path for the purpose of measuring object velocity, and to further employ improved signal processing, superior control system design, and/or a unique grating design to achieve high precision velocity measurements in an imaging flow cytometer.
The present invention is directed to a system and method for determining a velocity of an object. This method will preferably be employed in conjunction with an imaging or optical system that is adapted to determine one or more characteristics of an object from an image of the object or light from the object. There is relative movement between the object and the imaging system, and although it is contemplated that either, or both, the object and imaging system may be in motion, the object will preferably be in motion and the imaging system will be fixed. When used in conjunction with an imaging system that incorporates a time delay integration (TDI) detector, the velocity determined according to the present invention is used to provide a clocking signal to synchronize the TDI detector with the movement of the image over the TDI detector.
It should be understood that while portions of the following description, and the claims that follow, refer to xe2x80x9can object,xe2x80x9d it is clearly contemplated that the present invention is intended to be used with a plurality of objects, and is particularly useful in connection with imaging a stream of objects. In at least one embodiment, the stream of objects comprises moving cells or cell clusters. Furthermore, while the examples cited herein primarily relate to measuring the velocity of objects such as cells flowing in a stream of liquid, the present invention applies as well to objects attached to a solid substrate moving relative to the imaging system and to particles or liquid droplets flowing in a stream of gas.
The present invention involves the detection of light from an object and may optionally incorporate means for the illumination of the object. For the purpose of velocity measurement, the detected light must carry information about the change of position of the object over time. The object may modify the illumination light by scattering, refraction, diffraction, or absorption so that its wavelength is the same as that of the light received by the detector. Alternatively, fluorescence or phosphorescence at the object caused by the illumination light may result in light of different wavelengths than the illumination light to be received at the detector. Furthermore, the object may emit light used to determine the velocity of the object, without requiring prior illumination of the object by another light source.
The basic velocity measuring system includes a field of view (FOV) through which objects pass, preferably entrained in a fluid. Objects in the FOV may be illuminated, and light from the objects is collected to carry out the velocity measurement. The light from the object is modulated, producing modulated light having a frequency that depends upon the velocity of the object. The modulated light is detected, producing an electrical signal, and then the electrical signal is converted into a sequence of digital samples. The digitized signal is analyzed to extract the velocity of an object. In most embodiments, a light source is included to illuminate objects in motion. An optical grating modulates the light, and a light sensitive detector responds to the modulated light, producing a corresponding electrical signal that is digitized and processed to determine the velocity.
The light source may be a laser, a light emitting diode, a filament lamp, or a gas discharge arc lamp, for example, combined with optical conditioning elements such as lenses, apertures, and filters to deliver the desired wavelength(s) of light to the object with the intensity required for detection of the velocity. The light sensitive detector may be a photomultiplier tube or a solid-state photodetector and may be combined with optical conditioning elements such as lenses, apertures, and filters to deliver the desired wavelengths and direct the light from the object along a collection path and to the light sensitive detector.
At least one embodiment comprises a stage-based motion system with a high-resolution linear encoder, in which objects are deposited on a support, and the support is caused to move through the FOV. Regardless of whether the objects are entrained in a fluid or disposed on a support, the velocity of an object is determined with a Fast Fourier Transform (FFT) based signal processing system. In a preferred application, the processing system produces a frequency domain velocity measurement (FDVM) clocking signal that is useful in synchronizing a TDI detector to light from the object that is moving over the TDI detector.
Light from the object is received as the object passes through the FOV. Because the FOV is bounded by a profile of an illumination field and by an acceptance window of the light sensitive detector, it would be possible to estimate the velocity of the object from the time required for the object to pass through the FOV. However, the FOV is bounded by gradients rather than distinct edges, and it may be impractical to maintain the dimensions of the FOV with a high degree of accuracy.
To address the above-noted problem, one or more optical gratings is used to establish a distance measurement scale for computing velocity, eliminating the concern for maintaining the dimensions of the FOV with a high degree of accuracy. The optical gratings are preferably fabricated using high precision methods, such as photolithographic etching, to create patterns of alternating bars of opaque and transparent material. The accuracy required for the fabrication of the optical gratings is a function of the accuracy required in the velocity measurement. Preferably the optical grating includes alternating sequences of opaque strips and transparent strips of substantially equal width.
As noted above, the velocity measurement system can be used in conjunction with an imaging system that preferably employs a TDI detector. In TDI imaging, the charge in a TDI detector is moved in synchrony with an image that is incident on the TDI detector, enabling a many-fold increase in integration times over conventional frame imaging. TDI imaging is conventionally performed by clocking a pixel output from the detector using an encoder that directly measures sample movement, keeping the charge on the detector synchronized with the image. Since the transport of objects by a fluid stream prevents the use of an encoder, systems imaging objects in flow require an alternative means of synchronizing the TDI detector with the moving object and thus, with the moving image of the object across the TDI detector. The present invention provides an acceptable approach for measuring the velocity of moving objects (such as cells) for use in such TDI based imaging systems.
The present invention broadly encompasses two different preferred embodiments, including a first embodiment in which objects are disposed on a support that is moved through a sensitive or measurement volume, and a second embodiment in which objects are entrained in a flow of fluid, which is caused to flow through the sensitive or measurement volume. In each of these embodiments, optical gratings having a substantially uniform pitch are employed to modulate light received from the moving objects. The modulated light is converted into an electrical signal, which is digitized and then processed using a FFT to determine the velocity of the object. Preferably, each embodiment is executed under the control of a supervisory program.
A velocity measurement system in accord with the present invention includes an optical element disposed so that light traveling from an object that is passing through a FOV is directed along a collection path. An optical grating having a substantially uniform pitch is disposed in the collection path and produces modulated light having a modulation frequency proportional to a velocity of the object passing through the FOV. The pitch of the grating may be varied slightly to compensate for optical distortion by the optical element (e.g., a variation in magnification across the FOV), to produce a more consistent modulation frequency. A light sensitive detector is disposed in the collection path to receive the modulated light and convert the modulated light into an electrical signal. The system also includes means for converting the electrical signal into a sequence of digital samples and means for processing the sequence of digital samples, to determine the velocity of an object. The means for processing the sequence of digital samples utilizes a FFT function. In at least one embodiment, the means for processing the sequence of digital samples applies an amplitude windowing function to the sequence of numerical samples before applying the FFT function. The means for processing the sequence of digital samples can include a computer, an application specific integrated circuit, or a digital oscilloscope.
Preferably, the means for converting the electrical signal into a sequence of digital samples comprises an analog-to-digital converter. It is anticipated that most embodiments will also include an amplifier electrically coupled to the light sensitive detector, for amplifying the electrical signal before conversion of the electrical signal into a sequence of digital samples. In several embodiments, a bandpass filter is employed to filter the electrical signal before its conversion into a sequence of digital samples.
Preferably, the present invention will further include a system controller for controlling the acquisition of the electrical signal, for controlling the means for converting the electrical signal into a sequence of digital samples, and for controlling the means for processing the sequence of digital samples. The system controller will most preferably comprise a programmed computing device; however, it should be understood that an application specific integrated circuit (ASIC) or other hardwired logic circuit can also be beneficially employed as a system controller. Preferably the system controller includes means for controlling a gain of the amplifier in response to a magnitude of the electrical signal level that is coupled to the input of the amplifier from the light sensitive detector, means for determining a SNR of the electrical signal to preclude determining the velocity of the object passing through the FOV if the SNR is less than a predetermined minimum, and means for regulating a frequency range over which a mean frequency of the electrical signal from the detector is computed by the means for processing in response to variations in the velocity of an object passing through the FOV.
The light collected from an object passing through the FOV can include light scattered by that object, an unstimulated emission from the object, or a stimulated emission from the object. While it is contemplated that the velocity of the object can be measured using ambient light, preferably at least one light source is included for illuminating the FOV. Most preferably at least one light source is disposed to provide an incident light that illuminates an object passing through the FOV or to stimulate either an emission or a fluorescence from the object passing through the FOV. Also, the light from the source can be at least partially absorbed by an object passing through the FOV, changing the light from the object that passes through the optical element. Alternatively, the incident light is reflected from an object passing through the FOV toward the optical element. The light source can be one or more of a coherent light source, a noncoherent light source, a pulsed light source, a continuous light source, a continuous-wave laser, a pulsed laser, a continuous-wave incandescent lamp, a strobe arc lamp and an optical filter for selecting a limited spectrum for illumination.
Systems in accord with the present invention can beneficially incorporate a variety of different optical elements for directing light. In at least one embodiment the optical element is a lens, in another embodiment the optical element is a beam splitter, and in yet another embodiment the optical element is a dispersing element that directs the light from the object to each of at least two light-sensitive detectors.
Most preferably, the present invention is useful in measuring the velocity of objects entrained in a flow of fluid that is passing through the FOV. In other embodiments, the velocity of objects disposed on a support passing through the FOV can be measured. At least one embodiment comprises a stage-based motion system with a high-resolution linear encoder.
It is anticipated that velocity measuring systems, or combined velocity measurement and imaging systems, in accord with the present invention, can beneficially include a mechanism for sorting objects disposed downstream of the FOV. Regardless of the specific embodiment, the light sensitive detector can be either a photodiode, a photomultiplier tube, or other type of photodetector.
In at least one embodiment, the object passes through another FOV, and the system further includes another optical element disposed to direct light from the object passing through the other FOV along another collection path, and at least one additional light sensitive detector disposed to receive the light traveling along the other collection path and employed to determine a characteristic of the object passing through the other FOV.
Another embodiment of the present invention is directed to an optical analysis system employed to determine a velocity of a relative movement between an object and the optical analysis system, and at least one additional characteristic of the object. The optical analysis system includes a first optical element disposed to direct light from an object along a first collection path, a second optical element disposed in the first collection path to direct a portion of the light traveling from an object along the first collection path to a second collection path, and an optical grating of substantially uniform pitch disposed in the second collection path. The optical grating modulates the light traveling along the second collection path, thereby producing modulated light that has a modulation frequency proportional to a velocity of the relative movement between the object and the optical analysis system. This embodiment further includes a light sensitive detector disposed in the second collection path to receive the modulated light, the light sensitive detector producing an electrical signal in response to the modulated light. The system includes means coupled to the light sensitive detector to receive the electrical signal, for determining a velocity of the relative movement between the object and the optical analysis system as a function of the electrical signal using a Fast Fourier Transform (FFT) function, and for producing a timing signal as a function of the velocity. The system additionally includes a TDI detector disposed to receive light traveling along the first collection path, the TDI detector being coupled to the means for determining the velocity, so that the TDI detector can employ the timing signal to produce an output signal that is indicative of at least one nonvelocity characteristic of the object. Preferably, a control is coupled to, and controls the means for determining the velocity and the TDI detector.